In both the rat and human, prolactin (PRL) plays a pivotal role in mammary carcinogenesis. Results from many recent investigations suggest that the pituitary PRL "system" is a good deal more heterogeneous, and therefore complex, than previously suspected. At the molecular level, PRL variants, identified by Western blotting, are now known to be both contained in, and released from, PRL cells (mammotrophs). These variants have different biological/immunologic (B/I) activity ratios. Recent results from our laboratory demonstrate the probable association of unique variants with subpopulations of rat pituitary mammotrophs. Variants are also known to be contained in human plasma and media of human pituitary cells in vitro, but their role in mammary cancer remains undefined. Heterogeneity in structure and function of PRL-producing cells has also been uncovered using the techniques of cell separation and the reverse hemolytic plaque assay. Two new methods, described in this proposal, will permit assessment of mammotroph subpopulation heterogeneity in terms of secretory activity (B/I) by using 1-10 pituitary cells. Light scatter patterns and PRL/DNA fluorescent profiles obtained by flow cytometry will also permit definition of mammotroph heterogeneity both in terms of mammotroph number and mammotroph turnover. These "single cell" analyses will be done on cells contained within different regions (1/8) of a single gland. The hypothesis under test is that mammotrophs in different regions of the gland respond differently to agents suspected to ultimately lead to formation of pituitary and/or mammary tumor (E2,DMBA). FInally, the tissues of 1) functional heterogeneity of mammotrophs in surgical specimens of human pituitary tumors and 2) PRL variants in sera of breast cancer patients and women at risk because of their family history will also be studied. A second experimental objective relates to the production of a high MW protein from peritoneal macrophages which specifically inhibits PRL release from rat mammotrophs in vitro. Approaches to test this purified material in vivo are suggested. Since PRL is now known to modulate activity of cells in the immune system, identification of an active PRL inhibitor released from monocytes/macrophages would close the feedback loop of this as yet poorly defined endocrine-immune link.